VEGF165基因重组腺病毒载体的构建及其促血管形成作用

Construction and Expression of Recombinant Adenoviral Vector Expressing Human VEGF165

目的构建携带人血管内皮细胞生长因子165（VEGF165）基因的腺病毒表达载体（Ad—VEGF165），并观察其感染QBI．293A靶细胞后目的基因的表达及促进血管形成的作用。方法以含有VEGF165基因片段的pSV-K3-VEGF165质粒为模板，PCR扩增获得的VEGF165目的片段（XhoI、EcoRV）亚克隆至GFP标记的pAdTrack—CMV载体中构建重组转移质粒pAdTrack．CMV．VEGF165，行PCR、双酶切和DNA测序鉴定。将测序正确的pAdTrack—CMV．VEGFl65质粒经PmeI线性化后与腺病毒骨架质粒pAdEasy．1共转化BJ5183细菌，获得同源重组腺病毒质粒pAdEasy-1．pAdTrack-CMV．VEGF165（pAd．VEGF165），再经PacI线性化后用脂质体转染QBI．293A包装细胞，通过多轮扩增和氯化铯密度梯度离心纯化获得Ad．VEGFl65重组腺病毒。RT．PCR和ELISA检测腺病毒介导的外源性VEGFl65基因在QB｜．293A细胞中的转录和表达。将孵育8d的生长状态良好的鸡胚随机分成Ad空载体腺病毒组、Ad—VEGFI65重组腺病毒组、NS组。采用鸡胚尿囊膜（CAM）法检测VEGF165重组腺病毒促进尿囊膜血管形成活性。结果PCR、双酶切和测序鉴定结果证实成功构建了pAdTrack．CMV—VEGF165重组转移质粒，并获得了高滴度的Ad．VEGF165重组腺病毒，其病毒效价可达2×10^10pfu／mL。Ad-VEGFl65感染后，QBI．293A细胞中的VEGF165含量较QBI．293A细胞对照组和Ad空病毒感染组均明显升高（P〈0．05）。Ad-VEGF165组与Ad．空载体组和NS组比较，CAM三级分支血管生长更旺盛，毛细血管更丰富（P〈0．05）。结论成功获得了携带人VEGF165基因的腺病毒表达载体，具有明显的促CAM血管形成活性，为进一步开展VEGF165基因修饰干细胞的应用研究奠定了实验基础。

Objective To construct a recombinant adenoviral vector carrying and expressing human vascular endothelial cell growth factol： 165 （VEGF 165） （Ad-VEGF165） and examine the expression of VEGF165 in Ad- VEGF 165-infected QBI-293A target cells.Methods The VEGF 165 fragments were amplified by PCR using pSV- K3-VEGF165 plasmids as templates and subcloned into pAdTrack-CMV transfer vector at Xho I and EcoR V sites,which was identified by PCR, double endonuclease digestion and DNA sequencing.The pAdTrack-CMV-VEGF 165 transfer vector was linearized by Pme I digestion and pAdEasy-1 backbone vector were further cotransformed into the bacteria B J5183 cells for homologous recombination.The pAdEasy- 1-pAdTrack-CMV-VEGF 165 （pAd- VEGF 165） homologous recombinant plasmids were linearized with Pac I digestion and transfected into the human embryonic kidney 293 （QBI-293A） cells by Lipofectamine2000,1eading to the formation of the recombinant adenoviruses Ad-VEGF165.The Ad-VEGF 165 was amplified in QBI-293A cells and purified by CsCl density gradient centrifugation. Adenovirus-mediated transcription and expression of exogenous VEGF 165 gene in QBI-293A cells was examined by RT-PCR and ELISA respectively. 18 chick embryos hatched for 8 days were divided into 3 groups （Ad- group, Ad-VEGF165 and NS group at random）.The CAM assay was conducted to detect activity of VEGF165 recombinant adenovirus on promoting the angiogenesis of the chick chorioallantoic membrane.Results PCR, double endonuclease digestion and DNA sequencing showed that the VEGF165 fragments subcloned into pAdTrack-CMV transfer plasmids were completely identical to those reported in GenBank.We successfully ob- tained Ad-VEGF165 （2 × 10^10 pfu/mL） adenoviruses with high titre.Compared with QBI-293A cell control and Ad- infected QBI-293A blank adenovirus control group, Ad-VEGF 165-infected QBI-293A cells significantly transcribed and expressed higher levels ofVEGF 165 （P〈0.05）.Compared with the Ad group and NS group, the Ad-VEGF 165 gene group had a significant promoting effect on angiopoiesis of the chick chorioallantoic membrane. Conclusions The Ad-VEGF 165 adenoviral vector expressing VEGF 165 was successfully constructed,VEGF 165 recombinant adenovirus obviously promoted activity on the angiogenesis of the chick chorioallantoic membrane,which lays the experimental foundation for further research on VEGF 165 gene-modified stem cells.